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Abstract:

Provided is the use of an amphipathic weak base having defined
characteristics for the preparation of a pharmaceutical formulation for
the treatment or prevention of neurodegenerative conditions. The
amphipathic weak base can be encapsulated in a liposome. Also provided
are pharmaceutical formulations and methods of use thereof for the
treatment or prevention of neurodegenerative conditions. A specific and
amphipathic weak base is tempamine (TMN). Further, tempamine can be
loaded in sterically stabilized liposomes (SSL-TMN).

Claims:

1.-51. (canceled)

52. A method of treating a subject having, or in disposition of
developing, amyotrophic lateral sclerosis (ALS), the method comprising
administering to the subject a therapeutically effective amount of a
pharmaceutical formulation comprising an amphipathic weak base, the
amount being effective to treat or prevent the development of ALS,
wherein said amphipathic weak base has one or more of the following
characteristics: (i) it has pKa below 11.0; (ii) in an n-octanol/buffer
system having a pH of 7.0, it has a partition coefficient in the range
between 0.001 and 5.0; (iii) it exhibits an antioxidative activity; and
(iv) it exhibits a pro-apoptotic activity.

53. The method of claim 52, wherein the amphipathic weak base is
characterized by a pKa below 11.0 and a partition coefficient in the
range between 0.001 and 5.0.

54. The pharmaceutical formulation of claim 52, wherein the partition
coefficient is in the range of between 0.005 and 0.5.

62. A method of treating a subject having, or in disposition of developing
amyotrophic lateral sclerosis (ALS), the method comprising administering
to the subject a therapeutically effective amount of a pharmaceutical
formulation comprising tempamine, the amount being effective to treat or
prevent the development of ALS.

Description:

FIELD OF THE INVENTION

[0001]This invention generally concerns methods of treatment of
neurodegenerative conditions, in particular by using drugs encapsulated
by liposomes.

PRIOR ART

[0002]The following is the prior art which is considered to be pertinent
for describing the state of the art in the field of the invention.
[0003]WO03/053442; [0004]Nichols, J. W., et al., Biochim. Biophys. Acta
455:269-271 (1976); [0005]Cramer, J., et al., Biochemical and Biophysical
Research Communications 75(2):295-301 (1977).

BACKGROUND OF THE INVENTION

[0006]Neurodegenerative conditions, hereditary as well as sporadic
conditions, are characterized by progressive nervous system dysfunction.
These conditions are often associated with atrophy of the affected
central or peripheral nervous system structures.

[0008]Accumulating data indicate that oxidative stress (OS) plays a major
role in the pathogenesis of neurodegenerative diseases, such as MS,
through the generation of ROS primarily by macrophages. As a result,
demyelination and axonal damage are caused in both MS and experimental
autoimmune encephalomyelitis (EAE, the acceptable animal model for MS).

[0009]There are many attempts to develop antioxidants that can cross the
blood-brain barrier and decrease oxidative damage, leading to
neurodegenerative conditions. Natural antioxidants such as vitamin E
(tocopherol), carotenoids and flavonoids do not readily enter the brain
in the adult, and the lazaroid antioxidant tirilazad (U-74006F) appears
to localize in the blood-brain barrier. Thus, the use of modified spin
traps and low molecular mass scavengers of O2*.sup.- has been suggested
[Halliwell B. Drugs Aging. 18(9):685-716 (2001)].

[0010]In addition to overcoming the blood-brain barrier, the fast
clearance of antioxidants when administered in free form and their
chemical degradation in plasma limit their effectiveness in vivo. Thus, a
variety of approaches to extend the blood circulation time of these and
other therapeutic agents have been developed. One such approach included
the entrapment of the agent in a liposome.

[0011]There are a variety of drug-loading methods available for preparing
liposomes with entrapped drug, including passive entrapment and active
remote loading. The passive entrapment method is most suited for
entrapping of lipophilic drugs which reside in the liposome's membrane
and for entrapping drugs having high water solubility and/or high
molecular weight. However, this method of loading is limited by the
solubility of the drug in the hydration medium. In the case of ionizable
amphipathic drugs, even greater drug-loading efficiency can be achieved
by loading the drug into liposomes against a transmembrane ion gradient
[Nichols, J. W., et al., Biochim. Biophys. Acta 455:269-271 (1976);
Cramer, J., et al., Biochemical and Biophysical Research Communications
75(2):295-301 (1977)]. This loading method, generally referred to as
remote loading, typically involves a drug which is amphipathic and has an
ionizable amine group which is loaded by adding it to a suspension of
liposomes having a higher inside/lower outside H+ or ionizable
cation gradient (such as ammonium ions, for amphipathic weak bases) or
having a lower inside/higher outside H+ or ionizable anion gradient
(for amphipathic weak acids).

[0012]WO03/053442 describes a therapeutic formulation comprising tempamine
(TMN) for the treatment of conditions caused by oxidative stress or
cellular oxidative damage. The TMN is encapsulated in liposomes that
provide an extended blood circulation lifetime for the drug. TMN release
from liposomes, bio-distribution and pharmacokinetics of the liposome
entrapped TMN are described.

SUMMARY OF THE INVENTION

[0013]The present invention is based on several novel finding. Firstly, it
was found that tempamine (an amphipathic weak base antioxidant at times
referred to by the abbreviation, TMN) exhibits a protective effect on
PC12 neurons against 1-Methyl, 4-phenyl, Pyridinium ion (MPP+)
induced oxidative damage, and that the protective effect is in a dose
dependent manner.

[0014]Further, it was found that two different liposomal formulations
encapsulating, as the active ingredient, TMN, were significantly
effective in reducing clinical signs of multiple sclerosis (MS) and
Parkinson's disease (including incidence, duration and morbidity of the
disease), in acceptable animal models. In the experiments conducted,
sterically stabilized liposomes (SSL) encapsulating TMN SSL-TMN) were
used as TMN delivery system.

[0015]Yet further, it was found that the SSL-TMN formulations, having a
diameter of about 80 nm, were more effective in penetrating the blood
brain barrier (BBB) in experimental autoimmune encephalomyelitis (EAE,
the acceptable animal model for MS) as compared to their penetration
through the BBB of healthy animal.

[0016]Thus, it has been suggested that SSL-TMN may be of beneficial effect
against neurodegenerative disorders, particularly those requiring
penetration of a medication, through the blood brain barrier.

[0017]Thus, according to a first of its aspects, the present invention
provides the use of an amphipathic weak base for the preparation of a
pharmaceutical composition for the treatment or prevention of a
neurodegenerative condition, the amphipathic weak base having one or more
of the following characteristics: (i) it has pKa below 11.0; (ii) in an
n-octanol/buffer (aqueous phase) system having a pH of 7.0, it has a
partition coefficient in the range between about 0.001 and about 5.0,
preferably in the range between about 0.005 and about 0.5; (iii) it
exhibits an antioxidative activity; (iv) it exhibits a pro-apoptotic
activity.

[0018]In accordance with another aspect of the invention, there is
provided a pharmaceutical formulation for the treatment or prevention of
a neurodegenerative condition comprising as an active ingredient an
amphipathic weak base having one or more of the following
characteristics: (i) it has pKa below 11.0; (ii) in an n-octanol/buffer
(aqueous phase) system having a pH of 7.0, it has a partition coefficient
in the range between about 0.001 and about 5.0, preferably in the range
between about 0.005 and about 0.5; (iii) it exhibits an antioxidative
activity; (iv) it exhibits a pro-apoptotic activity.

[0019]In yet another aspect of the invention there is provided a method of
treating a subject having, or in disposition of developing a
neurodegenerative condition, the method comprising administering to said
subject an amount of pharmaceutical formulation comprising as active
ingredient an amount of an amphipathic weak base having one or more of
the following characteristics: (i) it has pKa below 11.0; (ii) in an
n-octanol/buffer (aqueous phase) system having a pH of 7.0, it has a
partition coefficient in the range between about 0.001 and about 5.0,
preferably in the range between about 0.005 and about 0.5; (iii) it
exhibits an antioxidative activity; (iv) it exhibits a pro-apoptotic
activity.

[0020]Preferably, the amphipathic weak base is characterized by at least
the above pKa and partition coefficient values.

[0021]The pharmaceutical composition should comprise a suitable
physiologically and pharmaceutically acceptable carrier. Typically the
carrier is such which allows the penetration of the active ingredient
thought the blood brain barrier (BBB). Such penetration is important
especially in neurodegenerative disease wherein the BBB remains
un-damaged.

[0022]The carrier may be a molecule which is known to promote or
facilitate entry through the BBB such as transferin receptor-binding
agents, antibodies, or any drug that by itself transfers through the BBB.
In such a case the molecule should be conjugated to the amphipathic weak
acid of the invention by a bond which is cleavable in the BBB.

[0023]Another alternative is to incorporate the active ingredient in a
suitable vehicle, such as lipid vesicles, nano-particles (coated or
uncoated) or nano-capsules, effective to penetrate the BBB.

[0024]By a preferred embodiment the active ingredient is encapsulated in a
lipid carrier, preferably a liposome as will be explained in more detail
below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]In order to understand the invention and to see how it may be
carried out in practice, a preferred embodiment will now be described, by
way of non-limiting example only, with reference to the accompanying
drawings, in which:

[0026]FIG. 1 is a bar graph showing TMN protection in PC12 neurons against
damage induced by MPP+. Cell death was evaluated by measuring the
leakage of lactic dehydrogenase (LDH) into the medium.

[0027]FIG. 2 is a graph showing the effect of sterically stabilized
liposomes loaded with TMN SSL-TMN) on clinical signs (clinical score) of
multiple sclerosis compared to that of commercially available drugs
(Copaxone, Betaferon), when using an EAE model of the disease. Saline was
used as control treatment.

[0029]FIG. 4A-4B are bar graphs showing the change in distribution of the
SSL-TMN liposomes in healthy (FIG. 4A) and EAE induced mice (FIG. 4B) in
the different tissues and in the plasma (plasma levels in FIG. 4A are
divided in two).

[0030]FIG. 5 is a graph showing the effect of SSL-TMN on clinical signs
(Mean clinical score) of multiple sclerosis compared to control treatment
(Saline) when using another EAE model of the disease.

[0031]FIG. 6 is a graph showing the effect of treatment with SSL-TMN on
6-OHDA Parkinson induced animal model.

[0032]FIG. 7 is a bar graph showing the behavioral change of animals
induced with 6-OHDA Parkinson after treatment with SSL-TMN (either i.v.
or s.c. injection) or with control (saline).

DETAILED DESCRIPTION OF THE INVENTION

[0033]The present invention concerns the use of an amphipathic weak base
encapsulated in a pharmaceutically acceptable drug delivery vehicle, to
form pharmaceutical formulations for treating neurodegenerative
conditions.

[0034]The term "amphipathic weak base" is used herein to denote a molecule
characterized by the following parameters: [0035](i) it has pKa below
11.0; preferably between about 11.0 and 7.5. [0036](ii) in an
n-octanol/buffer (aqueous phase) system having a pH of 7.0, it has a
partition coefficient in the range between about 0.001 and about 5.0,
preferably in the range between about 0.005 and about 0.5.

[0037]These above characteristics are described in length in WO03/053442
(Table 2), incorporated herein in its entirety by reference.

[0038]The amphipathic weak base is further characterized by its biological
activity, as an antioxidative agent and/or pro-apoptotic agent.

[0039]The term "antioxidant activity" or "antioxidative agent" refers to
the fact that the amphipathic weak base is capable of interacting with
free radicals, ROS and this are capable of preventing damage caused by
free radicals

[0040]The term "pro-apoptotic activity" or "pro-apoptotic agent" refers to
the fact that the amphipathic weak base is capable of inducing cell death
via the induction of apoptosis [as described in WO03/053442].

[0041]According to one embodiment, the amphipathic weak base is a
nitroxide compound. The term "nitroxide" is used herein to denote stable
cyclic nitroxide free radicals, their precursors and their derivatives
having a protonable amine, i.e. an amine capable of accepting at least
one hydrogen proton. Non-limiting examples of cyclic nitroxides include
carboxy nitroxides such as
5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO),
4-carboxy-2,2,6,6-tetramethylpiperidin-1-yloxyl (CTEMPO), and
3-carboxy-2,2,5,5-tetramethylpyrrodin-1-yloxyl (CPROXYL),
2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO),
4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL), and
-amino-2,2,2,6,6-tetramethyl-piperidine-N-oxyl (tempamine, TMN) A
preferred group of cyclic nitroxides are piperidine nitroxides. A
preferred amphipathic weak base in accordance with the invention which is
a piperidine nitroxide is TMN.

[0043]The term "neurodegenerative conditions" is used herein
interchangeably with the terms "neurodegenerative disease" and
"neurodegenerative disorder" to denote any abnormal deterioration of the
nervous system resulting in the dysfunction of the system. Further, it is
used to denote a group of conditions in which there is gradual, generally
relentlessly progressive wasting away of structural elements of the
nervous system exhibited by any parameter related decrease in neuronal
function, e.g. a reduction in mobility, a reduction in vocalization,
decrease in cognitive function (notably learning and memory) abnormal
limb-clasping reflex, retinal atrophy inability to succeed in a hang
test, an increased level of MMP-2, an increased level of neurofibrillary
tangles, increased tau phosphorylation, tau filament formation, abnormal
neuronal morphology, lysosomal abnormalities, neuronal degeneration,
gliosis and demyelination.

[0049]A preferred embodiment of the invention concerns the use of the
amphipathic weak base as characterized above (preferably such as
encapsulated in a liposome) for the preparation of a pharmaceutical
formulation for treatment of multiple sclerosis (MS).

[0050]Another preferred embodiment of the invention concerns the use of
the amphipathic weak base as characterized above (preferably such as
encapsulated in a liposome) for the preparation of a pharmaceutical
formulation for treatment of Parkinson's disease.

[0051]The terms "treat" or "treatment" are used herein to denote the
administering of a an amount of the amphipathic weak base encapsulated in
a pharmaceutically acceptable vehicle effective to prevent, inhibit or
slow down abnormal deterioration of the nervous system, to ameliorate
symptoms associated with a neurodegenerative condition, to prevent the
manifestation of such symptoms before they occur, to slow down the
irreversible damage caused by the chronic stage of the neurodegenerative
condition, to lessen the severity or cure a neurodegenerative condition,
to improve survival rate or more rapid recovery form such a condition. It
should be noted that in the context of the present invention the term
"treatment" also comprises prophylactic treatment i.e. for preventing
deterioration of the nervous system and thereby development of a
neurodegenerative conditions in subjects with high disposition of
developing a neurodegenerative condition (as determined by considerations
known to those versed in medicine) or for preventing the re-occurrence of
an acute stage of a neurodegenerative condition in a chronically ill
subjects. To this end, the vehicle loaded with the amphipathic weak base
may be administered to subjects who do not exhibit a neurodegenerative
condition but have a high-risk of developing such a condition, e.g. as a
result of exposure to an agent which may cause abnormal generation of
reactive oxidative species or subjects with family history of the disease
(i.e. genetic disposition). In this case, the vehicle loaded with the
amphipathic weak base will typically be administered over an extended
period of time in a single daily dose (e.g. to produce a cumulative
effective amount), in several doses a day, as a single dose for several
days, etc. so as to prevent the damage to the nervous system.

[0052]The term "effective amount" is used herein to denote the amount of
the amphipathic weak base when loaded in the vehicle in a given
therapeutic regimen which is sufficient to inhibit or reduce the
degradation of nerve cells and thereby the deterioration of the nervous
system. The amount is determined by such considerations as may be known
in the art and depends on the type and severity of the neurodegenerative
condition to be treated and the treatment regime. The effective amount is
typically determined in appropriately designed clinical trials (dose
range studies) and the person versed in the art will know how to properly
conduct such trials in order to determine the effective amount. As
generally known, an effective amount depends on a variety of factors
including the mode of administration, type of vehicle carrying the
amphipathic weak base, the reactivity of the amphipathic weak base, its
distribution profile within the body, a variety of pharmacological
parameters such as half life in the body after being released from the
vehicle, on undesired side effects, if any, on factors such as age and
gender of the treated subject, etc.

[0053]It is noted that humans are treated generally longer than
experimental animals as exemplified herein, which treatment has a length
proportional to the length of the disease process and active agent
effectiveness. The doses may be a single dose or multiple doses given
over a period of several days.

[0054]While the following disclosure provides experimental data with
animal model, there are a variety of acceptable approaches for converting
doses from animal models to humans. For example, calculation of
approximate body surface area (BSA) approach makes use of a simple
allometric relationship based on body weight (W) such that BSA is equal
to body weight (W) to the 0.67 power [Freireich E. J. et. al. Cancer
Chemother. Reports 1966, 50(4) 219-244; and as analyzed in Dosage Regimen
Design for Pharmaceutical Studies Conducted in Animals, by Mordenti, J,
in J. Pharm. Sci., 75:852-57, 1986]. Further, allometry and tables of BSA
data have been established [Extrapolation of Toxicological and
Pharmacological Data from Animals to Humans, by Chappell W & Mordenti J,
Advances in Drug Research, Vol. 20, 1-116, 1991 (published by Academic
Press Ltd)]

[0055]Another approach for converting doses is a pharmacokinetic-based
approach using the area under the concentration time curve (AUC) or
Physiologically Based PharmacoKinetic (PBPK) methods are described
[Voisin E. M. et al. Regul Toxicol Pharmacol. 12(2):107-116. (1990)]

[0056]The term "pharmaceutically/physiologically acceptable carrier" is
used herein to denote any acceptable vehicle suitable for delivery of an
active agent. Preferably it is a vehicle suitable to the delivery through
the BBB. The vehicle may be a lipid based vesicle (e.g. liposomes) or a
polymer based nanoparticle (e.g. where the polymer forms a matrix in
which the amphipathic weak base may be embedded or a shell structure,
where the amphipathic weak base is encapsulated within the core).
Preferably, the vehicle is a liposome. Further, preferably, the carrier
should be suitable for parenteral delivery of amphipathic weak bases,
specifically, for administration by injection. Other modes of
administration may include, without being limited thereto, oral,
intranasal (e.g. using a polycationic lipid-based liposomes such as CCS
described below), intra-ocular and topical administration as well as by
infusion techniques)

[0057]The term "liposome" is used herein to denote lipid based bilayer
vesicles. Liposomes are widely used as biocompatible carriers of drugs,
peptides, proteins, plasmic DNA, antisense oligonucleotides or ribozymes,
for pharmaceutical, cosmetic, and biochemical purposes. The enormous
versatility in particle size and in the physical parameters of the lipids
affords an attractive potential for constructing tailor-made vehicles for
a wide range of applications. Different properties (size, colloidal
behavior, phase transitions, electrical charge and polymorphism) of
diverse lipid formulations (liposomes, lipoplexes, cubic phases,
emulsions, micelles and solid lipid nanoparticles) for distinct
applications (e.g. parenteral, transdermal, pulmonary, intranasal and
oral administration) are available and known to those versed in the art.
These properties influence relevant properties of the liposomes, such as
liposome stability during storage and in serum, the biodistribution and
passive or active (specific) targeting of cargo, and how to trigger drug
release and membrane disintegration and/or fusion.

[0058]The liposomes are those composed primarily of liposome-forming
lipids which are amphiphilic molecules essentially characterized by a
packing parameter 0.74-1.0, or by a lipid mixture having an additive
packing parameter (the sum of the packing parameters of each component of
the liposome times the mole fraction of each component) in the range
between 0.74 and 1. Liposome-forming lipids, exemplified herein by
phospholipids, form into bilayer vesicles in water. The liposomes can
also include other lipids incorporated into the lipid bilayers, with the
hydrophobic moiety in contact with the interior, hydrophobic region of
the bilayer membrane, and the head group moiety oriented toward the
exterior, polar surface of the bilayer membrane.

[0059]The liposome-forming lipids are preferably those having a glycerol
backbone wherein at least one, preferably two, of the hydroxyl groups at
the head group is substituted with, preferably an acyl chain (to form an
acyl or diacyl derivative), however, may also be substituted with an
alkyl or alkenyl chain, a phosphate group or a combination or derivatives
of same and may contain a chemically reactive group, (such as an amine,
acid, ester, aldehyde or alcohol) at the headgroup, thereby providing a
polar head group. Sphyngolipids, such as sphyngomyelins, are good
alternative to glycerophopholipids.

[0060]Typically, the substituting chain(s), e.g. the acyl, alkyl or
alkenyl chain is between 14 to about 24 carbon atoms in length, and has
varying degrees of saturation being fully, partially or non-hydrogenated
lipids. Further, the lipid may be of natural source, semi-synthetic or
fully synthetic lipid, and neutral, negatively or positively charged.
There are a variety of synthetic vesicle-forming lipids and
naturally-occurring vesicle-forming lipids, including the phospholipids,
such as phosphatidylcholine (PC), phosphatidylinositol (PI),
phosphatidylglycerol (PG), dimyristoyl phosphatidylglycerol (DMPG); egg
yolk phosphatidylcholine (EPC), 1-palmitoyl-2-oleoylphosphatidyl choline
(POPC), distearoylphosphatidylcholine (DSPC), dimyristoyl
phosphatidylcholine (DMPC); phosphatidic acid (PA), phosphatidylserine
(PS) 1-palmitoyl-2-oleoylphosphatidyl choline (POPC) and the
sphingophospholipids, such as sphingomyelin (SM) having 12-24 carbon atom
acyl or alkyl chains. The above-described lipids and phospholipids whose
hydrocarbon chain (acyl/alkyl/alkenyl chains) have varying degrees of
saturation can be obtained commercially or prepared according to
published methods. Other suitable lipids include in the liposomes are
glyceroglycolipids and sphingoglycolipids and sterols (such as
cholesterol or plant sterol).

[0062]Cationic lipids (mono and polycationic) are also suitable for use in
the liposomes of the invention, where the cationic lipid can be included
as a minor component of the lipid composition or as a major or sole
component. Such cationic lipids typically have a lipophilic moiety, such
as a sterol, an acyl or diacyl chain, and where the lipid has an overall
net positive charge. Preferably, the head group of the lipid carries the
positive charge. Monocationic lipids may include, for example,
1,2-dimyristoyl-3-trimethylammonium propane (DMTAP)
1,2-dioleyloxy-3-(trimethylamino)propane (DOTAP);
N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimeth-yl-N-hydroxyethylammonium
bromide (DMRIE); N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy
ethyl-ammonium bromide (DORIE);
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA);
3β[N--(N',N'-dimethylaminoethane)carbamoly] cholesterol (DC-Chol);
and dimethyl-dioctadecylammonium (DDAB).

[0063]Examples of polycationic lipids include a similar lipophilic moiety
as with the mono cationic lipids, to which polycationic moiety is
attached. Exemplary polycationic moieties include spermine or spermidine
(as exemplified by DOSPA and DOSPER), or a peptide, such as polylysine or
other polyamine lipids. For example, the neutral lipid (DOPE) can be
derivatized with polylysine to form a cationic lipid. polycationic lipids
include, without being limited thereto,
N-[2-[[2,5-bis[3-aminopropyl)amino]-1-oxopentyl]amino]ethyl]-N,N-dimethyl-
-2,3-bis[(1-oxo-9-octadecenyl)oxy]-1-propanaminium (DOSPA), and ceramide
carbamoyl spermine (CCS).

[0064]The lipids mixture forming the liposome can be selected to achieve a
specified degree of fluidity or rigidity, to control the stability of the
liposome in serum and to control the rate of release of the entrapped
agent in the liposome.

[0065]Further, the liposomes may also include a lipid derivatized with a
hydrophilic polymer to form new entities known by the term lipopolymers.
Lipopolymers preferably comprise lipids, modified at their head group
with a polymer having a molecular weight equal or above 750Da. The head
group may be polar or apolar, however, is preferably a polar head group
to which a large (>750 Da) highly hydrated (at least 60 molecules of
water per head group) flexible polymer is attached. The attachment of the
hydrophilic polymer head group to the lipid region may be a covalent or
non-covalent attachment, however, is preferably via the formation of a
covalent bond (optionally via a linker). The outermost surface coating of
hydrophilic polymer chains is effective to provide a liposome with a long
blood circulation lifetime in vivo. The lipopolymer may be introduced
into the liposome by two different ways: (a) either by adding the
lipopolymer to a lipid mixture forming the liposome. The lipopolymer will
be incorporated and exposed at the inner and outer leaflets of the
liposome bilayer [Uster P. S. et al. FEBBS Letters 386:243 (1996)]; (b)
or by firstly prepare the liposome and then incorporate the lipopolymers
to the external leaflet of the pre-formed liposome either by incubation
at temperature above the Tm of the lipopolymer and liposome-30 forming
lipids, or by short term exposure to microwave irradiation.

[0066]Preparation of Vesicles Composed of Liposome-Forming Lipids and
Derivatization of such lipids with hydrophilic polymers (thereby forming
lipopolymers) has been described, for example by Tirosh et al. [Tirosh et
al., Biopys. J., 74(3):1371-1379, (1998)] and in U.S. Pat. Nos.
5,013,556; 5,395,619; 5,817,856; 6,043,094, 6,165,501, incorporated
herein by reference and in WO 98/07409. The lipopolymers may be non-ionic
lipopolymers (also referred to at times as neutral lipopolymers or
uncharged lipopolymers) or lipopolymers having a net negative or a net
positive charge.

[0068]While the lipids derivatized into lipopolymers may be neutral,
negatively charged, as well as positively charged, i.e. there is no
restriction to a specific (or no) charge, the most commonly used and
commercially available lipids derivatized into lipopolymers are those
based on phosphatidyl ethanolamine (PE), usually,
distearylphosphatidylethanolamine (DSPE).

[0069]A specific family of lipopolymers employed by the invention include
monomethylated PEG attached to DSPE (with different lengths of PEG
chains, the methylated PEG referred to herein by the abbreviated PEG) in
which the PEG polymer is linked to the lipid via a carbamate linkage
resulting in a negatively charged lipopolymer. Other lipopolymers are the
neutral methyl polyethyleneglycol distearoylglycerol (mPEG-DSG) and the
neutral methyl polyethyleneglycol oxycarbonyl-3-amino-1,2-propanediol
distearoylester (mPEG-DS) [Garbuzenko O. et al., Langmuir. 21:2560-2568
(2005)]. The PEG moiety preferably has a molecular weight of the head
group is from about 750 Da to about 20,000 Da. More preferably, the
molecular weight is from about 750 Da to about 12,000 Da and most
preferably between about 1,000 Da to about 5,000 Da. One specific
PEG-DSPE employed herein is that wherein PEG has a molecular weight of
2000 Da, designated herein .sup.2000PEG-DSPE or .sup.2kPEG-DSPE.

[0070]Preparation of Liposomes Including Such Derivatized Lipids has Also
been described, where typically, between 1-20 mole percent of such a
derivatized lipid is included in the liposome formulation.

[0071]As discussed above, the amphipathic weak base is preferably used in
combination with a vehicle. According to a preferred embodiment, the
vehicle is a lipid vesicle, and amphipathic weak base is encapsulated
within the vesicle. more preferably, the vesicle is a liposome.

[0072]The term "encapsulating" is used herein to denote the loading of the
amphipathic weak base into the aqueous phase of the lipid vesicle, e.g.
liposome. Loading is preferably achieved the use of remote loading
techniques where the antioxidant is loaded into pre-formed liposomes by
loading against an ammonium ion concentration gradient, as has been
described in U.S. Pat. No. 5,192,549. According to this method the
amphipathic weak base is accumulated in the intraliposome aqueous
compartment at concentration levels much greater than can be achieved by
other loading methods.

[0073]As used herein, "administering" is used to denote the contacting or
dispensing, delivering or applying the amphipathic weak base, preferably
carried by a vehicle, to a subject by any suitable route for delivery
thereof to the desired location in the subject, preferably by the
parenteral route including subcutaneous, intramuscular and intravenous,
intraarterial, intraperitoneally as well as by intranasal administration,
intrathecal and infusion techniques.

[0074]According to one preferred embodiment, the formulations used in
accordance with the invention are in a form suitable for injection. The
requirements for effective pharmaceutical vehicles for injectable
formulations are well known to those of ordinary skill in the art. See
Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co., Philadelphia,
Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook
on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986).

[0075]A preferred embodiment of the invention concerns liposomes
comprising between 1 to 20 mole percent of a lipopolymer. A preferred
hydrophilic moiety of the lipopolymer is PEG and a preferred derivatized
lipopolymer is either .sup.2000PEG-DSPE, .sup.2000PEG-DS or
.sup.2000PEG-DSG.

[0076]Variations in ratios between these liposome constituents dictate the
pharmacological properties of the liposome, including stability of the
liposomes, which is a major concern for various types of vesicular
applications. Evidently, the stability of liposomes should meet the same
standards as conventional pharmaceuticals. Chemical stability involves
prevention of both the hydrolysis of ester bonds in the phospholipid
bilayer and the oxidation of unsaturated sites in the lipid chain.
Chemical instability can lead to physical instability or leakage of
encapsulated drug from the bilayer and fusion and aggregation of
vesicles. Chemical instability also results in short blood circulation
time of the liposome, which affects the effective access to and
interaction with the target.

[0077]Specific liposomes compositions according to the invention are those
comprising a liposome forming lipid, such as hydrogenated soy
phosphatidylcholine (HSPC) or egg phosphatidylcholine (EPC), in
combination with cholesterol (Chol) and said lipopolymer. Specific
embodiments include the following liposome compositions:
EPC:Chol:.sup.2000PEG-DSPE and HSPC:Chol:.sup.2000PEG-DSPE both in a mole
ratio of 54:41:5. Evidently, other liposome forming lipids may be
utilized in the same or similar mole ratio, and provided that the final
additive packing parameter of the different constituents of the liposome
is in the range of between about 0.74 and 1.0.

[0078]According to a preferred embodiment of the invention pre-formed
liposomes are used for remote loading of the amphipathic weak base,
against an ion concentration gradient, into the liposomes. Liposomes
having an H+ and/or ion gradient across the liposome bilayer for use
in remote loading can be prepared by a variety of techniques. A typical
procedure comprises dissolving a mixture of lipids at a ratio that forms
stable liposomes in a suitable organic solvent and evaporated in a vessel
to form a thin lipid film. The film is then hydrated with an aqueous
medium containing the solute species that will form the intra-liposome
aqueous phase and will also serve the basis for the ion transmembrane
gradient (inner liposome high/outer medium low).

[0079]After liposome formation, the liposomes may be sized to achieve a
size distribution of liposomes within a selected range, according to
known methods. The liposomes are preferably uniformly sized to a selected
size range between 70-100 nm, preferably about 80 nm.

[0080]After sizing, the external medium of the liposomes is treated to
produce an ion gradient across the liposome membrane, which is typically
a higher inside/lower outside ion concentration gradient. This may be
done in a variety of ways, e.g., by (i) diluting the external medium,
(ii) dialysis against the desired final medium, (iii) gel exclusion
chromatography, e.g., using Sephadex G-50, equilibrated in the desired
medium which is used for elution, or (iv) repeated high-speed
centrifugation and resuspension of pelleted liposomes in the desired
final medium. The selection of the external medium will depend on the
mechanism of gradient formation, the external solute and pH desired, as
will now be considered.

[0081]In the simplest approach for generating an ion and/or H+
gradient, the lipids are hydrated and sized in a medium having a selected
internal-medium pH. The suspension of the liposomes is titrated until the
external liposome mixture reaches the desired final pH, or treated as
above to exchange the external phase buffer with one having the desired
external pH. For example, the original hydration medium may have a pH of
5.5, in a selected buffer, e.g., glutamate, citrate, succinate, fumarate
buffer, and the final external medium may have a pH of 8.5 in the same or
different buffer. The common characteristic of these buffers is that they
are formed from acids which are essentially liposome impermeable. The
internal and external media are preferably selected to contain about the
same osmolarity, e.g., by suitable adjustment of the concentration of
buffer, salt, or low molecular weight non-electrolyte solute, such as
dextrose or sucrose.

[0082]In another general approach, the gradient is produced by including
in the liposomes, a ion selective ionophore. To illustrate, liposomes
prepared to contain valinomycin in the liposome bilayer are prepared in a
potassium buffer, sized, then the external medium exchanged with a sodium
buffer, creating a potassium inside/sodium outside gradient. The K+
selective ionophore valinomycin enables movement of potassium ions in an
inside-to-outside direction in turn generates a lower inside/higher
outside pH gradient, presumably due to movement of protons into the
liposomes in response to the net electronegative charge across the
liposome membranes [Deamer, D. W., et al., Biochim. et Biophys. Acta
274:323 (1972)].

[0083]A similar approach is to hydrate the lipid and to size the formed
multilamellar liposome in high concentration of magnesium sulfate. The
magnesium sulfate gradient is created by dialysis against 20 mM HEPPES
buffer, pH 7.4 in sucrose. Then, the A23187 ionophore is added, resulting
in outwards transport of the magnesium ion in exchange for two protons
for each magnesium ion, plus establishing a inner liposome high/outer
liposome low proton gradient [Senske D B et al. (Biochim. Biophys. Acta
1414: 188-204 (1998)].

[0084]In another more preferred approach, the proton gradient used for
drug loading is produced by creating an ammonium ion gradient across the
liposome membrane, as described, for example, in U.S. Pat. Nos. 5,192,549
and 5,316,771, incorporated herein by reference. The liposomes are
prepared in an aqueous buffer containing an ammonium salt, such as
ammonium sulfate, ammonium phosphate, ammonium citrate, etc., typically
0.1 to 0.3 M ammonium salt, at a suitable pH, e.g., 5.5 to 7.5. The
gradient can also be produced by including in the hydration medium
sulfated polymers, such as dextran sulfate ammonium salt, heparin sulfate
ammonium salt or sucralfate. After liposome formation and sizing, the
external medium is exchanged for one lacking ammonium ions. In this
approach, during the loading the amphipathic weak base is exchanged with
the ammonium ion.

[0085]Yet, another approach is described in U.S. Pat. No. 5,939,096,
incorporated herein by reference. In brief, the method employs a proton
shuttle mechanism involving the salt of a weak acid, such as acetic acid,
of which the protonated form trans-locates across the liposome membrane
to generate a higher inside/lower outside pH gradient. An amphipathic
weak acid compound is then added to the medium to the pre-formed
liposomes. This amphipathic weak acid accumulates in liposomes in
response to this gradient, and may be retained in the liposomes by cation
(i.e. calcium ions)-promoted precipitation or low permeability across the
liposome membrane, namely, the amphipathic weak acid is exchanges with
the acetic acid.

[0086]The use of remote loading and in particular the latter ammonium ion
gradient procedure enables high loading of the amphipathic weak base into
the liposome. A preferred amphipathic weak base to lipid ratio is in the
range of between about 0.01 to about 2 and preferably between about 0.001
to about 4, preferably between 0.01 to about 2. For high loading of the
amphipathic weak base it is at times preferable that the concentration of
the same in the liposome be such that it precipitates in the presence of
a co-entrapped counter ion, such as sulfate.

[0087]According to another preferred embodiment, the loading of the
amphipathic weak base should be performed at a temperature range of the
gel to liquid crystalline phase transition.

[0088]The present invention preferably concerns the use of liposomal
formulations comprising a cyclic nitroxide as the amphipathic weak base.
A preferred amphipathic weak base is a cyclic nitroxide is TMN.

[0089]Thus, a preferred liposomal formulation according to the invention
is TMN encapsulated in sterically stabilized liposomes (SSL). In order to
penetrate at sufficient level the blood brain barrier, it is essential
that the SSL have a diameter of about 80 nm or smaller.

[0090]The following examples further illustrate the invention described
herein and are in no way intended to limit the scope of the invention.

[0093]Cell death was evaluated by measuring the leakage of LDH into the
growth medium as previously described [Abu-Raya et al. J. Neurosci. Res.
58:456-463, (1999)]. Samples of 50 μl of the growth medium were
collected from each well and centrifuged at 3,500 rpm for 5 min at
25° C.; the supernatant was collected and LDH release was measured
using a TRACE LD-L reagent. LDH activity was determined using an ELISA
reader (TECAN, SPECTRAFluor PLUS, Grodig, Salzburg, Austria) at 340 nm by
following the rate of conversion of oxidized nicotinamide adenine
dinucleotide (NAD+) to the reduced form of (NADH). MPP+-induced
LDH release was expressed as 100% of toxicity compared to
control-untreated cultures. Each experiment was performed three times in
duplicates (n=6).

MPP+ Toxicity Experiment

[0094]On the day of the experiment, the NGF containing medium was replaced
with fresh one. The cultures were divided into the three groups: 1)
control--untreated cells; 2) cultures exposed to MPP+insult; 3) TMN
treated cultures exposed to MPP+ insult.

[0095]MPP+ was dissolved in growth medium containing NGF and added to
each well in a final concentration of 1500 μM. At the end of
experiment, medium was taken for evaluation of LDH release. During the
experiment, all cultures were maintained in an incubator at 37° C.
in a humidified atmosphere of 6% CO2. The experiment was
accomplished when percentage of cell death was in the range 30-60%,
measured by the release of LDH into the medium.

[0096]TMN dissolved in growth medium containing NGF, was added to the
cultures 1 hr prior to the exposure to MPP+. For dose response
assay, TMN was administered to each well in a final concentrations of
0.1, 1, 10, 100, 500 or 1000 μM. Samples of 50 μl medium were taken
after 48 hr for assessment of LDH release.

[0097]FIG. 1 demonstrates that TMN protects PC12 neurons from oxidative
damage inflicted by 1500 μM MPP+ in a dose dependent manner in
the range of (0.1-100 μM), with 100 μM being most effective. The
bell shape at higher concentration 500 μM-1000 μM (irrelevant
concentrations for therapeutic applications) may imply that at higher
concentration TMN is toxic to the cells.

[0107]EPR spectrometry was employed to detect TMN concentration using a
JES-RE3X EPR spectrometer (JEOL Co., Japan) (Fuchs, J., et al., Free
Radic. Biol. Med. 22:967-976, (1997)). Samples were drawn by a syringe
into a gas-permeable Teflon capillary tube of 0.81 mm i.d. and 0.05 mm
wall thickness (Zeus Industrial Products, Raritan, N.J., USA). The
capillary tube was inserted into a 2.5-mm-i.d. quartz tube open at both
ends, and placed in the EPR cavity. EPR spectra were recorded with center
field set at 329 mT, 100 kHz modulation frequency, 0.1 mT modulation
amplitude, and nonsaturating microwave power. Just before EPR
measurements, loaded liposomes were diluted with 0.15 M NaCl for the
suitable TMN concentration range (0.02-0.1 mM). The experiment was
carried out under air, at room temperature. This is a functional assay
which determines the activity of TMN.

Cyclic Voltammetry (CV) Measurements

[0108]All cyclic voltammograms were performed between--200 mV and 1.3 V.
Measurements were carried out in phosphate-buffered saline, pH 7.4. A
three-electrode system was used throughout the study. The working
electrode was a glassy carbon disk (BAS MF-2012, Bioanalytical Systems,
W. Lafayette, Ind., USA), 3.3 mm in diameter. The auxiliary electrode was
a platinum wire, and the reference electrode was Ag/AgCl (BAS). The
working electrode was polished before each measurement using a polishing
kit (BAS PK-1) (Kohen, R., et al., Arch. Gerontol. Geriatr., 24:103-123,
(1996)). Just before CV measurements the samples were diluted with buffer
to the optimal TMN concentration range (0.05-0.2 mM). The experiments
were carried out under air, at room temperature. The CV assay is a
functional assay determining the ability of the analyte to accept or
donate electrons.

Liposome Preparation

Liposome Formation

[0109]A stock solution of EPC, Cholesterol and .sup.2000PEG-DSPE at a mole
ratio of 54:41:5 was mixed in ethanol at 70° C. to reach a final
lipid concentration of 62.5% (w/v), then incubated at 70° C. for
15 min until all the lipids were dissolved and a clear solution was
obtained. The ethanol stock solution containing lipid was then added to a
solution of 250 mM ammonium sulfate at 70° C. to reach a final
lipid concentration of 6.25% (w/v) reaching a final ethanol concentration
of 10% (w/v). The mixture was constantly stirred at 70° C. until a
milky dispersion was obtained, at this stage lipids were hydrated to form
un-sized heterogeneous multillamellar liposomes (MLVs).

[0110]Also the approach of lyophilization from tertiary buthanol (freezing
temperature of 22° C.) followed by mechanical hydration
(vortexing) and extrusion was used [G. Haran et al. Biochim Biophys. Acta
1151:201-215 (1993)]. All lipids were dissolved in tent-butanol and
lyophilized overnight. The dry lipid powder was hydrated with ammonium
sulfate solution (150 mM). Hydration was carried out above the Tm of
the matrix lipid: for HSPC, 60° C. (Tm=52.2° C.) and for
EPC room temperature, (Tm=-5° C.). Hydration was performed under
continuous shaking, forming multilamellar vesicles (MLV). The volume of
hydration medium was adjusted to obtain a 10% (w/v) lipid concentration.
Large unilamellar vesicles (LUV 100 nm) were prepared by stepwise
extrusion using a 100-nm-pore-size polycarbonate filter as the last step.

[0114]Liposome loading with TMN was performed as described in WO03/053442.
Briefly, a concentrated TMN alcoholic solution (0.8 ml of 25 mM TMN in
70% ethanol) was added to 10 ml of liposomal suspension. The final
solution contained 5.6% ethanol and 2 mM TMN. Loading was performed above
the Tm of the matrix lipid. Loading was terminated at the specified
time by removal of non-encapsulated TMN using the dialysis at 4°
C.

[0115]Loading efficiency was determined as described below.

Percent Encapsulation of Tempamine

[0116]The amount of entrapped TMN in liposomes prepared was determined as
described in WO03/053442 using either EPR or CV. For EPR measurements
first, the total TMN in the post-loading liposome preparation
(TMNmix) was measured. Then, the amount of TMN in the post-loading
liposome preparation in the presence of potassium ferricyanide, an EPR
broadening agent that eliminates the signal of free (non-liposomal) TMN,
was measured. The remaining signal was of TMN in liposomes
(TMNliposome(quenched)). The resulting spectrum was broad, as TMN
concentration inside the liposomes was high, leading to quenching of its
EPR signal due to spin interaction between the TMN molecules which are
close to one another. Then the total TMN after releasing it from
liposomes by nigericin (TMNnigericin) was measured. This signal was
identical to the total TMN used for loading
(TMNnigericin=TMNtotal) and is completely dequenched.
TMNliposome(not quenched) represents the signal of liposomal TMN
when the ammonium sulfate gradient is collapsed and all the TMN is
released.

[0117]The percent encapsulation and the quenching factor were calculated
as follows:

TMNfree=TMNmix-TMNliposomes(quenched) (1)

TMNliposomes(not quenched)=TMNnigericin-TMNfree (2)

Percent encapsulation=100×TMNliposome(not
quenched)/TMNnigericin (3)

Quenching factor=TMNliposome(not quenched)/TMNliposome(quenched)
(4)

[0118]The data are summarized in Table 1.

[0119]The level of TMN total=TMN nigericin agreed well with the TMN
determined after liposome solubilization by 1% Triton X-100. For TMN
determination by CV, firstly free TMN (remaining after loading into
liposomes) was determined. From these, level of free TMN, and percent TMN
encapsulated were calculated. There was a good agreement between EPR and
CV measurements as also described in WO03/053442.

[0120]TMN concentration in tissues, brain and plasma was quantified using
electron paramagnetic resonance (EPR) in the presence of 1.32% Triton
X-100 that solubilize the liposomes and enables detection of encapsulated
and free TMN levels, as described in the above methods section.

[0122]Free TMN (a concentrated TMN alcoholic solution (500 mM TMN in 70%
ETOH) was diluted in saline to obtain an 10 mM concentration or was added
to liposomes (EPC:Chol:.sup.2000PEG-DSPE) to reach a final concentration
of 10 mM TMN.

Liposomes Biodistribution:

[0123]Six to 7-week-SJL female mice, obtained through the Animal Breeding
House of the Hebrew University (Jerusalem, Israel), were used throughout
the biodistribution experiment. Animals were housed at Hadassah Medical
Center at an SPF faculty with food and water ad libitum. The experimental
procedures were in accordance with the standards required by the
Institutional Animal Care and Use Committee of the Hebrew University and
Hadassah Medical Organization.

[0125]Organs were homogenized in a Polytron homogenizer (Kinematica,
Lutzern, Switzerland) in 2% Triton X-100 (1:2, organ:Triton X-100
solution), cooled and heated several times to release the TMN. The plasma
samples were mixed 1:1 with 2% Triton X-100 to give the 1% Triton X-100
in the tested sample and also cooled and heated several times. Under such
conditions it was determined that intact liposomes released all their TMN
(for further TMN determinations).

[0126]Sample duplicates of 100 μl were burned in a Sample Oxidizer
(Model 307, Packard Instrument Co., Meridien, Conn.) left overnight in a
dark, cool place and measured by β-counting (KONTRON Liquid
Scintillation Counter). Radiospecific activity of the liposomes
DPM/μmole was calculated.

Example 2A

Multiple Sclerosis (MS)

Animal Model

A. Induction of Acute EAE Using PLP (Proteolipid Protein)

[0127]Induction of EAE using proteolipid protein was performed as
described in Pollak J of Neuroimmunology 137:94-99 (2003)]. In brief, 6-7
week old SJL female mice were immunized by subcutaneous injection in the
right flank with an emulsion containing proteolipid protein (PLP) 139-151
peptide and complete Freund's adjuvant (CFA) containing 150 μg of
peptide and 200 μg of Mycobacterium tuberculosis. On the day of the
first PLP injection, Pertussis Toxin (PT) 150 ng was injected
intraperitoneally (0.1 ml/mice).

[0130]Once clinical signs of MS appeared (i.e. on day 10 post inoculation
with PLP), the mice received treatment either with a conventional MS
medication such as Betaferon (Schering AG Germany) or Copaxone (Teva
pharmaceuticals, Israel), or with the sterically stabilized TMN
formulation (EPC:Chol:.sup.2000PEG-DSPE, 54:41:5, SSL-TMN in Table 2
below) described in Table 1 above.

[0131]The mice were observed daily from the 10th day post-EAE induction
(PLP injection, i.e. the first day of treatment) and the EAE clinical
signs were scored. The scores were performed according to Table 3 below:

TABLE-US-00003
TABLE 3
clinical signs scoring
Score Signs Description
0 Normal behavior No neurological signs
1 Distal limp tail The distal part of the tail is limp and droops
1.5 Complete limp tail The whole tail is loose and droops
2 Complete limp tail with The whole tail is loose and droops. Animal has
righting reflex difficulties to return on his feet when it is laid on his
back
3 Ataxia Woobly walk-when the mouse walks the hind legs
are unsteady
4 Early paralysis The mouse has difficulties standing on its hind legs
but still has remnants of movement
5 Full paralysis The mouse can't move its legs at all, it looks thinner
and emaciated. Incontinence
6 Moribund/death

[0132]The number of mice in each animal group which developed the disease
(sick) was summed and the percentage thereof was calculated.

[0133]In addition, the mean maximal score (MMS) by summing the maximal
scores of each of the 10 mice in the group and calculating therefrom the
mean maximal score of the group according to the following equation:

Σmaximal score of each mouse/number of mice in the group

[0134]Further, the mean duration of disease (MDD) expressed in days was
calculated according to the following equation:

Σduration of disease of each mouse/number of mice in the group

[0135]Further, each group's mean score (GMS) (burden of disease) was
determined by summing the scores of each of the 10 mice in the group and
calculating the mean score per day, according to the following equation:

Σtotal score of each mouse per day/number of mice in the group.

[0136]Tables 4A, 4B and 4C (obtained from three separate assays) and FIG.
1 summarize the different scores calculated:

[0137]The results above and in FIG. 2 demonstrate that intravenous
administration of sterically stabilized TMN SSL-TMN, 80 nm in diameter)
was more effective in reducing the clinical signs of MS as compared to
the signs observed with conventional medications (Copaxone and Betaferon)
or as compared to empty SSL liposomes (EPC or HSPC) or free TMN, the
empty liposomes or free TMN having no observed effect against the
disease.

[0139]Organ samples were homogenized in a Polytron homogenizer
(Kinematica, Lutzern, Switzerland) in 2% Triton X-100 (1:2, organ:Triton
X-100 solution), cooled and heated several times to destroy the lipid
membrane. The plasma samples were mixed 1:1 with 2% Triton X-100 to give
the 1% Triton X-100 in the tested sample and also cooled and heated
several times. It was determined that under such conditions intact
liposomes released all their TMN content.

[0140]Sample duplicates of 100 μl were burned in a Sample Oxidizer
(Model 307, Packard Instrument Co., Meridien, Conn.) left overnight in a
dark, cool place and measured by β-counting (KONTRON Liquid
Scintillation Counter), reflecting the amount of liposomal TMN in each
organ. FIG. 3 presents the percent of absorbance per ml tissue in healthy
and EAE induced mice, after treatment with liposomal TMN
(EPC:Chol.sup.2000PEG-DSPE). Specifically shown is that [3H]
Cholesteryl hexadecyl ether SSL-TMN liposomes penetration was higher in
brains of diseased (EAE) mice than in that of healthy mice, particularly
during the first 6 hours after injection of [3H] Cholesteryl
hexadecyl ether SSL-TMN liposomes. It is assumed that this is a result of
a disruption in the blood brain barrier (BBB) which is common with MS and
similar neurodegenerative disorders.

[0141]The difference in tissue distribution of the liposomal TMN in
healthy and diseases animal models is shown in FIG. 4A-4B respectively.

[0142]Induction of chronic EAE using MOG 35-55 peptide was performed as
described in [Offen D et al J Mol Neurosci. 15(3):167-76 (2000)]. In
general, female C57B1/6 mice were inoculated (s.c. injection in the right
flank) with an encephalitogenic emulsion (MOG plus CFA enriched with MT
(mycobacterium tuberculosis). Pertussis toxin was injected i.p (250
ng/mouse) on the day of inoculation and 48 hrs later. A boost of the MOG
emulsion was injected s.c. in the right flank one week after first
injection. On day 10, each mouse was injected (i.v.) with SSL-TMN
formulation or with the control solution. The animals were kept in SPF
conditions and given food and water ad libitum. Treatment was terminated
on day 30.

[0143]For treatment, the animals (10 mice per group) were divided into
groups and treated as summarized in Table 5 below.

[0144]The mice were observed daily from the 10th day post-EAE induction
(first injection of MOG) and the EAE clinical signs were scored according
to the Table 3 shown above. The results are presented in Table 6 and FIG.
5.

[0145]Table 6 and FIG. 5 show that SSL-TMN was effective in reducing the
clinical signs of MS also in MOG induced animal model of the chronic
disease as compared to the control (saline)

Example 2B

Parkinson Disease

[0146]For determining the effect of the liposomal TMN formulation in
treating Parkinson disease the conventional 6-Hydroxydopamine (6-OHDA)
Parkinson animal model was used [Hastings T G et al; Proc. Natl. Acad.
Sci. USA 93:195619-195661 (1996)]. This model is characterized by the
unilateral injection of 6-OHDA into the substantia nigra with the
ulterior accumulation of the toxin (6-OHDA) into dopaminergic neurons
leading to their death presumably mediated by oxidative stress. In brief,
6-OHDA (8 μg/rat) was stereotaxically injected in 4 μl into the
right substantia nigra of male Sprague-Dawley rats (weighing 250-280 g;
coordinates of injection: P=4.8, L=1.7, H=-8.6 from bregma). Eighteen
days after 6-hydroxydopamine injection, rats were selected for
transplantation if they had >350 rotations per hour after s.c.
injection of apomorphine (25 mg/100 g body weight) and, if 2 days later,
they also had >360 (mean 520±38) rotations per hour after i.p.
injection of D-amphetamine (4 mg/kg).

[0147]The effectiveness of the lesion in the substantia nigra was
evaluated with the stepping test [Olsson M et al; J. neurosci
15(5):3863-3875 (1995)]. This test determines motor initiation deficits
in the forelimbs of the rats, analogous to limb akinesia and gait problem
in Parkinson patients The 6-OHDA lesion profoundly affect the adjusting
steps, it means that there is a significant impairment in the left paw
performance (contralateral to the lesion) which results in a dragging paw
when the rat is moved sideways by the experimenter. By contrast right paw
is unaffected. Animals receiving SSL-TMN (EPC:Chol:.sup.2000PEG-DSPE)
show a significant increase in the adjusting steps number in contrast
with the control 6OHDA animals The number of stepping adjustments was
counted for each forelimb during slow sideway movements in forehand
directions over a standard flat surface. The stepping adjustments test
was performed twice for each forelimb after SSL-TMN injection and the
SSL-TMN treated animals restored the number of adjusting steps to a level
similar from that seen in intact control animals (animals that didn't
receive 6-OHDA).The stepping test was repeated at least three times
between days 15 and 20 after the lesion in all the rats. Only those rats
treated with 6-OHDA and which showed less than two adjusting steps with
the forelimb contralateral to the lesion during each trial were selected
for treatment.

[0148]Specifically, rats were divided into two groups:

[0149]Group I--rats receiving treatment with 1 ml SSL-TMN (either i.v. or
s.c. injection) 2 and 4 days before induction of the disease with 6-OHDA

[0150]Group II--rats receiving treatment with 1 ml SSL-TMN 2, 4 and 7 days
after the induction of the disease with 6-OHDA.

[0151]The rats were observed daily from the day of induction (day 0), and
the clinical signs were scored. Results are presented in FIG. 6.

[0152]The behavior of the rats was also examined through the stepping test
described above. Specifically, the percent of improvement in the stepping
adjustment test (left paw over the right paw ×100) was scored, the
results of which are shown in FIG. 7.

Patent applications by Haim Ovadia, Jerusalem IL

Patent applications by Pablo Kizelsztein, Yishuv Lapid IL

Patent applications by Yechezkel Barenholz, Jerusalem IL

Patent applications by HADASIT MEDICAL RESEARCH SERVICES & DEVELOPMENT LIMITED

Patent applications by YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM